Gemcitabine protide hypoxia-activated prodrug and application thereof

ABSTRACT

A gemcitabine ProTide hypoxic-activated prodrug and a use thereof in the preparation of a medicament for treating tumors. The general structural formula thereof is formula (A), wherein: one of R1 and R2 is a hypoxic-activated group of —C(R3R4)ArNO2, and the other is an alkyl group of 1 to 6 carbon atoms, a phenyl group or —CH2Ar, wherein R3 and R4 are —H or a methyl group, and —Ar is an aromatic ring compound. The gemcitabine ProTide hypoxic-activated prodrug described in the present invention has a stronger cytotoxicity under a hypoxic condition, has excellent anti-tumor effects and is very safe; the present invention can be used along with other anti-tumor drugs to exert a better anti-tumor activity, and can be used in the preparation of a medicament for treating tumors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national phase entry of International Application No. PCT/CN2017/095794, filed on Aug. 3, 2017, which is based upon and claims priority to Chinese Patent Application No. 201610649914.X, filed on Aug. 9, 2016, and Chinese Patent Application No. 201710610509.1, filed on Jul. 25, 2017, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention belongs to the field of pharmacy, and provides a gemcitabine ProTide hypoxic-activated prodrug and a use thereof.

BACKGROUND

Gemcitabine is a nucleoside anti-tumor drug. The mechanism of action of this drug is to antagonize nucleotide metabolism. After intracellular triphosphorylation in vivo, gemcitabine specifically interferes with nucleic acid metabolism and prevents cell division and reproduction by inhibiting the synthesis of deoxynucleoside triphosphate (dNTPs), interfering with cell replication by being incorporated into DNA or RNA molecules, competitively inhibiting DNA polymerase, and the like, thus eventually causing the death of tumor cells. Nucleoside anti-tumor drugs are prone to drug resistance, but ProTide prodrugs thereof can reduce the occurrence of drug resistance and have a good anti-tumor effect, among which a gemcitabine ProTide prodrug NUC-1031 has been clinically studied (Journal of Medical Chemistry 2014, 57, 1531-1542). However, ProTide prodrugs cannot reduce the toxic and side effects of drugs on non-tumor tissues.

With the rapid growth of tumors, some tumor tissues are farther and farther away from the nearest blood vessel, and oxygen supply is insufficient, resulting in tumor hypoxia (Nature review cancer 2002, 2: 38-47). Traditional anti-tumor drugs have good lethality to tumors near blood vessels, but have limited effects on tumors in hypoxic regions. Tumor hypoxic-activated prodrugs can specifically release anti-tumor active constituents in tumor hypoxic regions, thus killing tumors in the hypoxic regions (Chinese Journal of Cancer 2014, 33: 80-86). Hypoxic-activated prodrugs have a tumor targeting property, thus having a better safety performance, and a better anti-tumor effect when used in combination with traditional anti-tumor drugs, among which TH302 has been clinically studied and has a good therapeutic effect on pancreatic cancer (Journal of Clinical Oncology 2015, 33, 1475-1482).

SUMMARY Technical Problem

The present invention provides a gemcitabine ProTide hypoxic-activated prodrug and a use thereof. The prodrug has a stronger cytotoxicity under a hypoxic condition, has excellent anti-tumor effects and is very safe, can be used along with traditional anti-tumor drugs to exert a good anti-tumor activity at a small dose, and can be used in the preparation of a medicament for treating tumors.

Technical Solution

The chemical structural formula of the gemicitabine ProTide hypoxic-activated prodrug is:

wherein one of R¹ and R² is a hypoxic-activated group of —C(R³R⁴)ArNO₂, the other is an alkyl group of 1 to 6 carbon atoms, a phenyl group or —CH₂Ar, R³ and R⁴ are —H or a methyl group, and —Ar is an aromatic ring compound.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activated prodrug, the structure of R¹ is

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ is —H or a methyl group, and R⁴ is a methyl group.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activated prodrug, R¹ is a phenyl group, and the structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activated prodrug, the structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ and R⁴ are —H.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activated prodrug, R¹ is —CH₂Ar, —Ar is a benzene ring with an electron donating group, and the structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.

As a preferred scheme, the structure of the gemcitabine ProTide hypoxic-activated prodrug is as follows:

As a preferred scheme, the structure of the gemcitabine ProTide hypoxic-activated prodrug is as follows:

A use of the above compound or pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors.

A use of a composition of the above compound or pharmaceutically acceptable salt thereof and gemcitabine hydrochloride in the preparation of a medicament for treating tumors.

A medicament for treating tumors, of which the effective component being the above gemcitabine ProTide hypoxic-activated prodrug or pharmaceutically acceptable salt thereof.

A medicament for treating tumors, of which the effective component being the composition of the above gemcitabine ProTide hypoxic-activated prodrug or pharmaceutically acceptable salt thereof and gemcitabine hydrochloride.

It should be pointed out that our research found that the cytotoxicity of compounds 001-021 under a hypoxic condition was significantly higher than that under a normal oxygen condition. The cytotoxicity of a target compound (e.g., compound 022) obtained by introducing a hypoxic-activated group into an amino position of gemcitabine ProTide prodrug NUC-1031 under a hypoxic condition was not significantly different from that under a normal oxygen condition (see Table 1). It indicated that the introduction position of the hypoxic-activated group was specific for maintaining the hypoxic-activated function of drugs.

It should also be pointed out that our research found that when R² was a hypoxic-activated group, if R³ and R⁴ were both H (such as compound 023), the cytotoxicity of the target compound under a hypoxic condition was not significantly different from that under a normal oxygen condition (see Table 1), while when R¹ was a hypoxic-activated group, if R³ and R⁴ were both H (such as compounds 013-017), the cytotoxicity of the target compound under a hypoxic condition was significantly higher than that under a normal oxygen condition (see Table 1).

When R¹ was a hypoxic-activated group, R² was —CH₂Ar, and Ar was a phenyl group with an electron donating group, the target compound showed a stronger cytotoxicity under a hypoxic condition (such as compounds 018-021); when Ar was a phenyl group having no substituent group or having an electron with-drawing group (such as compound 024), the cytotoxicity of the target compound under a hypoxic condition differed slightly from that under a normal oxygen condition.

Taking compound 001 as an example, the anti-tumor effect and safety performance of the target compound were investigated. The target compound showed a significant anti-tumor growth effect (see FIG. 1 and FIG. 2). At 4 times of treatment dose, compared with a control group, there was no significant difference in animal weight, indicating that the target compound had good safety performance (see FIG. 3). Combined use with traditional anti-tumor drugs such as gemcitabine can generate better anti-tumor effect (see FIG. 2).

Advantageous Effect

The gemcitabine ProTide hypoxic-activated prodrug of the present invention has a small cytotoxicity in a normal oxygen environment and strong cytotoxicity under a hypoxic condition, therefore, the gemcitabine ProTide hypoxic-activated prodrug can specifically play an anti-tumor effect on tumors in tumor hypoxic regions, reduce toxic and side effects on other tissues, has an excellent anti-cancer effect and good safety performance, can be used together with traditional anti-tumor drugs such as gemcitabine to generate a good anti-tumor effect at a small dose, and can be used for preparing medicaments for treating tumors. Further research finds that at 4 times of effective dose, the gemcitabine ProTide hypoxic-activated prodrug provided by the present invention has no obvious toxic effect increase compared with low dose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a growth inhibition effect of a target compound 001 on orthotopic transplantation tumor of human BxPC-3 nude mice. After administration, the pancreatic cancer tumor tissue quality of nude mice in an experimental group (compound 001) was significantly lower than that of a gemcitabine group and an NUC-1031 group, indicating a better tumor growth inhibition effect.

FIG. 2 is a schematic diagram of a growth inhibition effect of a target compound 001 on subcutaneous orthotopic transplantation tumor of human BxPC-3 nude mice. After administration, the pancreatic cancer tumor tissue volume of nude mice in a high-dose group and a low-dose+gemcitabine group was significantly lower than that of a gemcitabine group, indicating a better tumor growth inhibition effect.

FIG. 3 is a schematic diagram of changes in animal weight when a target compound 001 is used for treating subcutaneous orthotopic transplantation tumor of human BxPC-3 nude mice. Compared with a low-dose group (1 time of the effective dose), the animal weight of a high-dose group (4 times of the effective dose) is not significantly different from that of the low-dose group and a positive control drug gemcitabine group, indicating that the target compound has good safety performance.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following embodiments enable those skilled in the art to fully understand the present invention, but do not limit the present invention in any way.

Embodiment 1: Synthesis of Target Compounds 001-021:

Synthesis of 3′-O-(t-butyloxycarboryl) gemcitabine

Synthesis route (method reference, The Journal of Organic Chemistry, 1999, 64: 8319-8322):

Experimental operation: stir gemcitabine (0.60 g, 2 mmol), Na₂CO₃ (1.06 g), 40 mL dioxane, 40 mL water, di-tert-butyl dicarbonate ester (DBDC, 0.44 g, 2 mmol) at room temperature for 48 h; add 20 mL; water, extract with 2×300 mL ethyl acetate, dry with Na₂SO₄, and perform vacuum concentration; perform flash column chromatography on (CH₂Cl₂-ethyl acetate-EtOH 1:1:0.02) to obtain 3′-O-(N-t-butyloxycarboryl) gemcitabine (0.60 g). ¹H NMR (DMSO-d6, 300 MHz) δ(ppm): 7.64 (d, 1H) 7.40 (d, 2H) 6.21 (t, 1H), 5.81 (d, 1H), 5.25-5.12 (m, 2H), 4.13 (t, 1H) 3.71-3.60 (m, 2H), 1, 45 (s, 9H).

Synthesis route (method reference, The Journal of Medicinal Chemistry, 2014, 57:1531-1542):

Add 10 mL redistilled dichloromethane into a 100 mL eggplant shape flask, add phosphorus oxychloride (0.2 g, 1.3 mmol) into dichloromethane, place the reaction system at −78° C., add triethylamine (0.13 g, 1.3 mmol) therein, stir for 15 min, then dropwise add a dichloromethane solution (5 mL) of phenol (0.153 g, 1 mmol), react at −78° C. for 1 h after 15 min of dropping, and react at room temperature for 1 h; leave the whole reaction system at −78° C., add 0.23 g (2.3 mmol) triethylamine therein, stir for 15 min, add 10 ml redistilled dichloromethane solution containing 0.275 g (1 mmol) L-alanine-1-(4-nitrophenyl) ethanol ester hydrochloride, and stir for 3 h; add 30 mL redistilled dichloromethane into another 50 mL eggplant shape flask, add 0.29 g (0.8 mmol) 3′-O-(t-butyloxycarboryl) gemcitabine, add 0.2 g (2 mmol) triethylamine, add 2 mL N-methylimidazole, and add the dichloromethane solution into the 100 mL reaction system in the previous flask at room temperature for overnight reaction at room temperature; concentrate the solvent, filter out insoluble substances, wash the filtrate with 3×30 mL water, extract with dichloromethane, concentrate the solvent, stir with 6 mL trifluoroacetic acid and dichloromethane (volume ratio being 1:1) at 0° C. for 4 h, concentrate the solvent, and perform flash column chromatography (volume ratio of dichloromethane: methanol being 20:1); and obtain the compound 001: ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.22-8.21 (m, 2H), 7.60-7.55 (m, 1H), 7.52-7.51 (m, 2H), 7.41-7.36 (m, 2H), 7.29-7.22 (m, 3H), 6.30-6.25 (m, 1H), 6.04-5.89 (m, 2H), 4.56-4.38 (m, 2H), 4.28-4.21 (m, 1H), 4.15-4.10 (m, 1H), 3.97-3.91 (m, 1H), 1.56-1.55 (m, 3H), 1.38-1.32 (m, 3H).

Compounds 002-021 were synthesized with the same method.

Referring to the method for producing the compound 001, phenol, L-alanine-1-(4-nitrophenyl)-1-methyl ethanol ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.21-8.20 (m, 2H), 7.65-7.64 (m, 2H), 7.54-7.50 (m, 1H), 7.35-7.30 (m, 2H), 7.23-7.18 (m, 3H), 6.25-6.19 (m, 1H), 5.99-5.85 (m, 1H), 4.48-4.37 (m, 2H), 4.23-4.14 (m, 1H), 4.09-4.04 (m, 1H), 3.91-3.84 (m, 41-1), 1.74-1.73 (m, 6H), 1.32-1.26 (m, 3H).

Referring to the method for producing the compound 001, phenol, L-alanine-1-(3-methyl-2-nitro-3H-imidazole-4-yl) ethanol ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 Hz) δ(ppm): 7.58-7.53 (m, 1H), 7.40-7.33 (m, 2H), 7.30-7.22 (m, 3H), 7.22-7.20 (m, 1H), 6.26-6.23 (m, 1H), 6.02-5.90 (m, 1H), 5.85-5.75 (m, 1H), 4.59-4.41 (m, 2H), 4.20-4.30 (m, 1H), 397-390 (m, 4H), 1.60-1.59 (m, 3H), 1.34-1.27 (m, 3H).

Referring to the method for producing the compound 001, phenol, L-alanine-1-(5-nitro-furan-2-yl) ethanol ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 7.69 (d, 1H), 7.57-7.52 (m, 1H), 7.38-7.33 (m, 2H), 7.26-7.19 (m, 3H), 6.93 (d, 1H), 6.27-6.21 (m, 1H), 5.97-5.90 (m, 1H), 4.53-4.31 (m, 2H), 4.25-4.18 (m, 1H), 4.12-4.07 (m, 1H), 3.94-3.87 (m, 1H), 1.57-1.56 (m, 3H), 1.35-1.29 (m, 3M),

Referring to the method for producing the compound 001, phenol, L-alanine-1-(5-nitro-thiophene-2-yl) ethanol ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.75 (s, 1H), 7.72 (s, 1H), 7.55-7.50 (m, 1H), 7.36-7.31 (m, 2H), 7.24-7.18 (m, 3H), 6.26-6.21 (m, 1H), 6.07-6.01 (m, 1H), 5.90-5.81 (m, 1H), 4.51-4.30 (m, 2H), 4.24-4.16 (m, 1H), 4.10-4.04 (m, 1H), 3.92-3.86 (m, 1H), 1.60-1.59(m, 3H), 1.32-1.27 (m, 3H).

Referring to the method for producing the compound 001, 1-(4-nitrophenyl) ethanol, L-alanine benzyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeO), 300 Hz) δ(ppm): 8.20-8.18 (m, 2H), 7.64-7.62 (m, 2H), 7.58-7.53 (m, 1H), 7.39-7.34 (m, 5H), 6.28-6.23 (m, 1H), 5.92-5.76 (m, 2H), 5.16-5.10 (m, 2H), 4.54-4.32 (m, 2H), 4.26-4.20 (m, 1H), 4.12-4.06 (m, 1H), 3.95-3.90 (m, 1H), 1.56-1.53 (m, 3H), 1.35-1.30 (m, 3H).

Referring to the method for producing the compound 001, -(4-nitrophenyl) ethanol, L-alanine methyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.22-8.20 (m, 2H), 7.67-7.65 (m, 2H), 7.60-7.55 (m, 1H), 6.31-6.26 (m, 1H), 5.96-5.80 (m, 2H), 4.57-4.35 (m, 2H), 4.29-4.23 (m, 1H), 4.15-4.09 (m, 1H), 3.97-3.92 (m, 1H), 3.58 (s, 3H), 1.61-1.55 (m, 3H), 1.38-1.33 (m, 3H).

Referring to the method for producing the compound 001, 1-(4-nitrophenyl) ethanol, L-alanine ethyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.19-8.17 (m, 2H), 7.63-7.61 (m, 2H), 7.58-7.52 (m, 1H), 6.26-6.21 (m, 1H), 5.90-5.75 (m, 2H), 4.52-4.30 (m, 2H), 4.24-4.18 (m, 1H), 4.14-4.05 (m, 1H), 3.91-3.75 (m, 3H), 1.55-1.50 (m, 3H), 1.34-1.29 (m, 3H), 1.16-1.12 (m, 3H).

Referring to the method for producing the compound 001, 1-(4-nitrophenyl) ethanol, L-alanine isopropyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.21-8.19 (m, 2H), 7.65-7.63 (m, 2H), 7.60-7.56 (m, 1H), 6.29-6.25 (m, 1H), 5.94-5.88 (m, 2H), 5.03-4.97 (m, 1H), 4.55-4.34 (m, 2H), 4.28-4.22 (m, 1H), 4.15-4.10 (m, 1H), 3.98-3.93 (m, 1H), 1.58-1.54 (m, 3H), 1.38-1.31 (m, 3H), 1 26-1.23 (m, 6H).

Referring to the method for producing the compound 001, 1-(4-nitrophenyl) ethanol, 1,-alanine butyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.23-8.21 (m, 2H), 7.65-7.63 (m, 2H), 7.62-7.57 (m, 1H), 6.31-6.25 (m, 1H), 5.97-5.79 (m, 2H), 4.58-4.35 (m, 2H), 4.30-4.24 (m, 1H), 4.17-4.12(m, 1H), 4.00-3.95 (m, 1H), 3.88-3.78 (m, 2H), 1.60-1.20 (m, 10H), 0.85-0.81 (m, 3H).

Referring to the method for producing the compound 001, 1-(4-nitrophenyl) ethanol, L-alanine 2,2-dimethyl propyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ^(1H) NMR (MeOD, 300 MHz) δ(ppm): 8.24-8.22 (m, 2H), 7.66-7.64 (m, 2H), 7.62-7.58 (m, 1H), 6.32-6.27 (m, 1H), 5.99-5.80 (m, 1H), 4.60-4.38 (m, 2H), 4.32-4.26 (m, 1H), 4.18-4.14 (m, 1H), 4.02-3.96 (m, 1H), 3.90-3.80 (m, 2H), 1.62-1.58 (m, 3H), 1.39-1.33 (m, 3H), 1.24-1.21 (m, 9H).

Referring to the method for producing the compound 001, 1-(4-nitrophenyl) ethanol, L-alanine cyclohexyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.19-8.17 (m, 2H), 7.63-7.61 (m, 2H), 7.57-7.52 (m, 1H), 6.28-6.23 (m, 1H), 5.94-5.78 (m, 2H), 4.55-4.35 (m, 2H), 4.28-4.20 (m, 2H), 4.15-4.10 (m, 111), 4.02-3.94 (m, 1H), 1.70-1.52 (m, 5H), 1.39-1.15 (m, 11H).

Referring to the method for producing the compound 001, 1-(5-nitrothiophene-2-yl) methanol, L-alanine cyclohexyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.08-8.07 (m, 1H), 7.65-7.55 (m, 1H), 7.32-7.31 (m, 1H), 6.30-6.25 (m, 1H), 5.98-5.88 (m, 1H), 5.25-5.07 (m, 3H), 4.55-4.35 (m, 1H), 4.28-4.21 (m, 1H), 4.15-4.10 (m, 1H), 4.04-3.97 (m, 1H), 1,72-1.55 (m, 5H), 1.42-1.18 (m, 11H).

Referring to the method for producing the compound 001, 4-nitrobenzyl alcohol, L-alanine methyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.20-8.19 (m, 2H), 7.65-7.63 (m, 2H), 7.59-7.53 (m, 1H), 6.28-6.24 (m, 1H), 5.95-5.90 (m, 1H), 5.14-5.02 (m, 2H), 4.55-4.35 (m, 2H), 4.28-4.21 (m, 1H), 4.15-4.10 (m, 1H), 3.98-3.90 (m, 1H), 3.56 (s, 3H), 1.57-1.53 (m, 3H), 1.36-1.30 (m, 3H).

Referring to the method for producing the compound 001, 1-(5-nitrofuran-2-yl) methanol, L-alanine benzyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ (ppm): 7.69-7.68 (m, 1H), 7.65-7.64 (m, 1H), 7.59-7.54 (m, 1H), 7.36-7.29 (m, 5H), 6.29-6.24 (m, 1H), 5.94-5.88 (m, 1H), 5.08-5.03 (m, 2H), 4.57-4.36 (m, 2H), 4.30-4.20 (m, 1H), 4.14-4.09 (m, 1H), 3.97-3.88 (m, 1H), 3.89-3.60 (m, 2H), 1.56-1.51 (m, 3H), 1.35-1.31 (m, 3H).

Referring to the method for producing the compound 001, 1-(5-nitrothiophene-2-yl) methanol, L-alanine isopropyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.07-8.06 (m, 2H), 7.67-7.55 (m, 1H), 7.31-7.300, 1H), 6.30-6.25 (m, 1H), 5.96-5.88 (m, 1H), 5.22-5.18 (m, 2H), 5.09-4.98(m, 4.56-4.35 (m, 1H), 4.32-4.21 (m, 1H), 4.15-4.10 (m, 1H), 3.99-3.90 (m, 1H), 1.38-1.33 (m, 3H)1.26-1.24 (m, 6H).

Referring to the method for producing the compound 001, (3-methyl-2-nitro-3H-imidazole-4-yl) methanol, L-alanine ethyl ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 7.66-7.55 (m, 1H), 7.22-7.21 (m, 1H), 6.29-6.23 (m, 1H), 5.94-5.86 (m, 1.1H), 4.54-4.31 (m, 2H), 4.26-4.18 (m, 1H), 4.17-4.10 (m, 3H), 3.96-3.70 (m, 3H), 1.36-1.32 (m, 3H), 1.16-1.13 (m, 3H).

Referring to the method for producing the compound 001, 4-methoxybenzyl alcohol, L-alanine-1-methyl-1-(5-nitrofuran-2-yl) ethanol ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 7.68-7.66 (m, 1H), 7.61-7.56 (m, 1H), 7.05-7.04 (m, 2H), 6.96-6.94 (m, 2H), 6.32-6.25 (m, 1H), 5.96-5.87 (m, 2H), 5.10-5.03 (m, 2H), 4.57-4.34 (m, 2H), 4.29-4.21 (m, 1H), 4.15-4.09 (m, 1H), 3.98-3.91 (m, 4H), 1.56-1.55 (m, 3H), 1.37-1.32 (m, 3H).

Referring to the method for producing the compound 001, 2-methyl benzyl alcohol, L-alanine-1-methyl-1-(5-nitrothiophene-2-yl) ethanol ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.73-8.72 (m, 1H), 7.64-7.63 (m, 1H), 7.59-7.54 (m, 1H), 7.37-7.32 (m, 1H), 7.24-7.15 (m, 3H), 6.31-6.23 (m, 1H), 5.95-5.89 (m, 1H), 5.13-5.00 (m, 2H), 4.56-4.32 (m, 2H), 4.28-4.10 (m, 3H), 4.14-4.06 (m, 1H), 3.97-3.90 (m, 1H), 2.37-2.35 (m, 3H), 1.82-1.81 (m, 6H), 1.37-1.30 (m, 6H).

Referring to the method for producing the compound 001, 4-N,N-dimethylbenzyl alcohol, L-alanine-1-methyl-1-(5-nitrothiophene-2-yl) ethanol ester hydrochloride, gemcitabine and other raw materials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm):8.20-8.18 (m, 2H), 7.61-7.56 (m, 1H), 7.50-7.49 (m, 2H), 7.06-6.83 (m, 4H), 6.30-6.21 (m, 1H), 6.02-5.89 (m, 2H), 5.11-4.98 (m, 2H), 4.56-4.32 (m, 2H), 4.29-4.19 (m, 1H), 4.12-4.06 (m, 1H), 3.96-3.88 (m, 1H), 2.81-2.80 (m, 6H), 1.82-1.81 (m, 6H), 1.35-1.29 (m, 3H).

Referring to the method for producing the compound 001, 2-methoxybenzyl alcohol, L-alanine-1-(3-methyl-2-nitro-3H-imidazole-4-yl) ethanol ester hydrochloride, gemcitabine and other raw materials were used for synthesis, ¹H NMR (MeOD, 300 MHz) δ(ppm): 7.55-7.61 (m, 1H), 7.35-7.25 (m, 2H), 7.00-6.92 (m, 2H), 7.20-7.19 (m, 1H), 6.26-6.23 (m, 1H), 5.96-5.84 (m, 1H), 5.04-4.90 (m, 2H), 4.58-4.40 (m, 2H), 4.28-4.17 (m, 1H), 3.95-3.88 (m, 411), 1.59-1.58 (m, 3H), 1.30-1.20 (m, 3H).

Embodiment 2: Research on In-Vitro Inhibitory Effect of Target Compound on Tumor Cell Proliferation Under Normal Oxygen State and Hypoxic State

Take tumor cells in the logarithmic growth phase, add 0.25% pancreatin for digestion for 3 min, use RPMI-1640 containing 10% calf serum for suspension culture of the cells, count the number, adjust cell concentration to 1×10⁵ cells/mL, inoculate to a Top-count dedicated 96-well cell culture plate at 100 μL/well, and incubate at 37° C. and 5% CO₂ for 24 h; divide the cells into experimental groups and control groups, and add a target compound solution (0.001 μg/mL, 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL) to the experimental groups, wherein each concentration corresponded to four wells, and the volume of each well was made up to 200 μL; after adding samples, continue to culture for 72 for each group (for hypoxic groups, continue to culture for 72 h at 5% CO₂, 95% N₂), add ³H-TdR 3×10⁵Bq to each group before culture ended, and measure the CPM (count per minute) value of each well with Top-count; and calculate the median inhibition concentration (IC₅₀) of drugs in each experimental group on cell proliferation.

TABLE 1 Median inhibition concentration (IC₅₀, μ/mL) of target compound on tumor cell proliferation (72 h) under normal oxygen and hypoxic conditions IC₅₀ normal oxygen (air)/IC₅₀ hypoxic (nitrogen) Human lung Human liver Human pancreatic Compound adenocarcinoma cancer adenocarcinoma cell number cell A549 HepG2 cell BxPC-3 cell 001  1/0.05 >10/0.3 >10/0.2 002 2/0.1 >10/0.2 >10/0.4 003 Not measured >10/0.4 Not measured 004 Not measured Not measured >10/0.4 005 2/0.1 Not measured Not measured 006 2/0.2 Not measured >10/0.4 007 Not measured >10/0.5 >10/0.4 008 Not measured >10/0.3 >10/0.4 009 2/0.2 Not measured >10/0.4 010 2/0.2 Not measured Not measured 011 2/0.2 Not measured Not measured 012 Not measured >10/0.5 >10/0.5 013 Not measured Not measured >10/0.8 014 Not measured Not measured >10/0.7 015 Not measured Not measured >10/0.5 016 Not measured Not measured >10/0.4 017 Not measured Not measured >10/0.8 018 Not measured Not measured >10/0.5 019 Not measured Not measured >10/0.8 020 Not measured Not measured >10/0.4 021 Not measured Not measured >10/0.5 022 0.01/0.01   0.4/0.3  0.1/0.1 023 0.01/0.01   0.4/0.3  0.1/0.1 024 Not measured Not measured  >10/>10 Gemcitabine 0.01/0.01   0.2/0.2  0.2/0.2 NUC-1031 0.01/0.01   0.2/0.2  0.1/0.1

The above experimental results show that gemcitabine, NUC-1031 and compounds 022-024 have no significant difference in in-vitro inhibition on tumor cell proliferation under normal oxygen and hypoxic conditions, and compounds (1-021) of the embodiments of the present invention have significant difference (10-50 times) in in-vitro inhibition on tumor cell proliferation under normal oxygen and hypoxic conditions, indicating that the compounds of the embodiments of the present invention have a stronger cytotoxicity for tumors in hypoxic regions.

Embodiment 3: Growth Inhibition Effect of Target Compound on Orthotopic Transplantation Tumor of Human BxPC-3 Nude Mice

Take BxPC-3 human pancreatic cancer cells in the logarithmic growth phase, inoculate subcutaneously on the back of nude mice at a concentration of 5×10⁶ cells·0.2 mL⁻¹·mouse⁻¹, establish a human BxPC-3 nude mice subcutaneous transplantation tumor model, take out after growing into a 1 cm subcutaneous transplantation tumor, remove the central necrotic tissue under an aseptic condition, and select and cut the surrounding healthy tumor tissue into 1 mm³ tissue blocks.

Preparation of surgical orthotopic transplantation model: intraperitoneally anesthetize nude mice with pentobarbital sodium (50 mg/Kg), make a cut beside the left upper rectus abdominis muscle to expose spleen and tail of pancreas, cut open capsula pancreatis, implant a tumor block into the tail of pancreas near splenic artery, and suture the capsula pancreatis.

Administration scheme: model animals were randomly divided into an experimental group (compound 001), a control group, a gemcitabine group and an NUC-1031 group 3 weeks after operation, from the third week after operation, the nude mice were injected intraperitoneally (0.2 mmol/kg, twice per week) for 4 weeks, the nude mice were killed one week after drug withdrawal, and pancreatic tumor tissues were taken and weighed. See FIG. 1 for inhibition effect: growth inhibition effect of target compound on orthotopic transplantation tumor of human BxPC-3 nude mice. After administration, the pancreatic tumor tissue quality of nude mice in the experimental group (compound 001) was significantly lower than those of the gemcitabine group and the NUC-1031 group, indicating a better tumor growth inhibition effect.

Embodiment 4: Growth Inhibition Effect of Target Compound on Subcutaneous Transplantation Tumor of Human BxPC-3 Nude Mice

Take BxPC-3 human pancreatic cancer cells in the logarithmic growth phase, inoculate subcutaneously on the back of nude mice at a concentration of 5×10⁶ cells·0.2 mL⁻¹·mouse⁻¹, three weeks later, after the long diameters of the transplanted tumors in nude mice were all ≥5 mm, calculate the similar volume of tumor bodies based on the long diameter and short diameter of the transplanted tumors. Nude mice were divided into 5 groups by a random block design and allocation method according to the tumor volume.

Administration scheme: 50 model animals were randomly divided into a negative control group, a low-dose group (compound 001, 0.1 mmol/kg), a high-dose group (compound 001, 0.4 mmol/kg), a gemcitabine hydrochloride group (0.2 mmol/kg), and a combined administration group (compound 001, 0.1 mmol/kg+gemcitabine hydrochloride, 0.1 mmol/kg), intraperitoneally injected (twice per week) for 3 weeks, and killed one week after drug withdrawal. At the same time, animal weight was measured and the eye condition of the animal was observed.

See FIG. 2 for inhibition effect and FIG. 3 for weight change: after administration, each group showed a significant tumor growth inhibition effect, and the high-dose group and the combined administration group showed a better therapeutic effect. The weights of nude mice in all experimental groups had no significant difference, but were smaller than those of the control groups.

The above examples are only to illustrate the technical concept and features of the present invention, with the purpose of enabling those familiar with the technology to understand the content of the present invention and implement it accordingly, but do not limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be included within the scope of protection of the present invention. 

What is claimed is:
 1. A gemcitabine ProTide hypoxic-activated prodrug, wherein a chemical structural formula of the gemcitabine ProTide hypoxic-activated prodrug is:

wherein one of R¹ and R² is a hypoxic-activated group of —C(R³R⁴)ArNO₂, the other of R¹ and R² is an alkyl group of 1 to 6 carbon atoms, a phenyl group or —CH₂Ar, R³ and R⁴ are —H or a methyl group, and —Ar is an aromatic ring compound.
 2. The gemcitabine ProTide hypoxic-activated prodrug according to claim 1, wherein a structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ is —H or a methyl group, and R⁴ is a methyl group.
 3. The gemcitabine ProTide hypoxic-activated prodrug according to claim 1, wherein R¹ is a phenyl group, a structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.
 4. The gemcitabine ProTide hypoxic-activated prodrug according to claim 1, wherein a structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ and R⁴ are —H.
 5. The gemcitabine ProTide hypoxic-activated prodrug according to claim 1, wherein R¹ is —CH₂Ar, —Ar is a benzene ring with an electron donating group, a structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.
 6. The gemcitabine ProTide hypoxic-activated prodrug according to claim 1, wherein the chemical structural formula of the gemcitabine ProTide hypoxic-activated prodrug is one selected from the group consisting of chemical structural formulas as follows:


7. The gemcitabine ProTide hypoxic-activated prodrug according to claim 1, wherein the chemical structural formula of the gemcitabine ProTide hypoxic-activated prodrug is one selected from the group consisting of chemical structural formulas as follows:


8. A method of preparing a medicament for treating tumors, comprising using a compound according to claim 1 or pharmaceutically acceptable salt of the gemcitabine ProTide hypoxic-activated prodrug.
 9. A medicament for treating tumors, wherein an effective component of the medicament for treating tumors is the gemcitabine ProTide hypoxic-activated prodrug according to claim 1 or pharmaceutically acceptable salt of the gemcitabine ProTide hypoxic-activated prodrug.
 10. The medicament according to claim 9, wherein a structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ is —H or a methyl group, and R⁴ is a methyl group.
 11. The medicament according to claim 9, wherein R¹ is a phenyl group, a structure of R² is:

R³ is —H or a methyl group, and is a methyl group.
 12. The medicament according to claim 9, wherein a structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ and R⁴ are —H.
 13. The medicament according to claim 9, wherein R¹ is —CH₂Ar, —Ar is a benzene ring with an electron donating group, a structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.
 14. The medicament according to claim 9, wherein the chemical structural formula of the gemcitabine ProTide hypoxic-activated prodrug is one selected from the group consisting of chemical structural formulas as follows:


15. The medicament according to claim 9, wherein the chemical structural formula of the gemcitabine ProTide hypoxic-activated prodrug is one selected from the group consisting of chemical structural formulas as follows:


16. The method of claim 8, wherein a structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ is —H or a methyl group, and R⁴ is a methyl group.
 17. The method of claim 8, wherein R¹ is a phenyl group, a structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.
 18. The method of claim 8, wherein a structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ and are —H.
 19. The method of claim 8, wherein R¹ is —CH₂Ar, —Ar is a benzene ring with an electron donating group, a structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.
 20. The method of claim 8, wherein the chemical structural formula of the gemcitabine ProTide hypoxic-activated prodrug is one selected from the group consisting of chemical structural formulas as follows: 